Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel V_HH nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1) associated with high T-cell polyfunctionality. This study demonstrates the potential of V_HH-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes.

异质抗原表达是影响实体瘤中嵌合抗原受体（CAR）T细胞活性的关键障碍。在这里，我们开发了针对磷脂酰肌醇蛋白聚糖-1 (GPC1) 的 CAR T 细胞，这是一种在胰腺癌中表达的癌胎儿抗原。我们报道了单峰骆驼 V _H H 纳米抗体 (D4) 的靶向 GPC1 的 CAR T 细胞的生成，以及铰链 (H) 和跨膜结构域 (TM) 的优化以提高活性。我们发现，结构刚性的 IgG4H 和 CD28TM 结构域使两个 D4 片段靠近，驱动 CAR 二聚化，并导致雌性小鼠抗原密度低的胰腺癌模型中 T 细胞信号传导增强和肿瘤消退。此外，D4-IgG4H-CD28TM CAR T细胞的基于单细胞的蛋白质组学和转录组学分析揭示了与高T细胞多功能性相关的特定基因（例如， HMGB1 ）。这项研究证明了基于_VHH的 CAR T 在胰腺癌治疗中的潜力，并为开发靶向膜远端表位的有效 CAR T 细胞提供了工程策略。